The mechanism for malignancy of mammalian cells has been and continues to be the subject of intense investigation. One of the most promising areas is the elucidation of how oncogenes are turned on and turned off.
Oncogenes were first detected in retroviruses, and then cellular counterparts of the viral oncogenes were found. A score of oncogenes has been isolated since the early 1970's from retroviruses that variously cause carcinoma, sarcoma, leukemia or lymphoma in chickens, other birds, rats, mice, cats or monkeys. In each case, the oncogene has been found to be closely related to a normal gene in the host animal and to encode an oncogenic protein similar to a normal protein. The proteins encoded by oncogenes function abnormally and seem to play a part in ordaining the transformation of a normal cell into a cancer cell. Genes in the DNA of various kinds of tumor cells, when introduced by transfection into normal cultured cells, transform the normal cells into cancer cells.
The oncogenes are virtual or almost virtual copies of proto-oncogenes. Whatever the specific mechanism that converts a proto-oncogene into an oncogene may be, an oncogene exerts its effect by means of the protein it encodes. The products of the proto-oncogenes appear to have roles that are critical in the regulation of cell growth and differentiation and in embryonic development. Transforming proteins may have their profound effects on cells because they disturb these fundamental cellular processes.
Enzymatic activity in catalyzing the addition of a phosphate molecule to an amino acid (phosphorylation) is known to be important in the control of protein function. The enzymes that phosphorylate proteins are called protein kinases (from the Greek kinein, "to move"). Almost one-third of all the known oncogenes code for protein kinases specific for tyrosine residues.
Epidermal growth factor (EGF), when added to a culture of individual cells, stimulate the cells to divide. EGF in vitro can trigger a variety of morphological and biochemical changes that resemble those characteristic of transformed cells, and has also been implicated in the abnormal regulation of proliferation shown by transformed and tumor-derived cell lines. Epidermal growth factor (EGF) promotes the growth of many cell types in vitro and inhibits proliferation of several cell types, for example, A431 epidermoid carcinoma cells and certain human breast cancer cells [Kawamoto et al., PNAS (USA), 80: 1337-1341 at p. 1337 (March 1983)].
EGF delivers its signals by binding to specific protein receptors embedded in the cell's plasma membrane. When the receptor protein for EGF was isolated, it was found to be associated with tyrosine kinase activity, which is stimulated when an EGF molecule binds to the receptor.
The receptor protein for EGF, the epidermal growth factor receptor (EGFr), is a 170 kilodalton (kd) transmembrane glycoprotein which comprises a cytoplasmic or tyrosine kinase domain, a transmembrane region and an extracellular domain or ectodomain which contains the binding site for epidermal growth factor (EGF) and transforming growth factor alpha (TGF-.alpha.) [Marquardt and Todaro, J. Biol. Chem., 257: 5220-5225 (1982); Carpenter et al., PNAS (USA) 80: 5627-5630 (1983)]. The cytoplasmic domain comprises 542 amino acids (C-terminal residues), and the ectodomain comprises 621 amino acids (N-terminal residues); they are linked by a short transmembrane region of about 23 amino acids [Ullrich et al., Nature, 309 (5967): 418-425 (May 31, 1984); Gullick et al., Proc. R. Soc. Lond., B 226: 127-134 (1985)].
EGFr is a cell membrane macromolecule that is widely distributed in cells derived from all three embryonic germ layers. The wide distribution of the receptor in cells and tissues indicates that EGF may have an important role to play in growth control. Many normal cells express 10-100,000 EGFrs [Adamson and Rees, Mol. Cell Biochem., 34: 129-152 (1981); however, increased numbers of EGFrs are present in several types of human tumors, including gliomas and meningiomas, squamous carcinoma of the lungs, and ovarian, cervical and renal carcinomas [Thompson and Gill, Cancer Surveys, 4(4): 768-788 (1985]. The human epidermoid carcinoma cell line A431 expresses about 10- to 50-fold more receptors than the majority of other cells [Fabricant et al., PNAS (USA), 74(2): 565-569 (1977); Wrann and Fox, J. Biol. Chem., 254: 8083-8086 (1979)].
The gene that encodes the EGFr is related to the avian erythroblastosis virus (AEV) oncogene which encodes the v-erb-B transforming protein. The v-erb-B oncogene is highly homologous with the EGFr transmembrane and tyrosine kinase domains; however, the v-erb-B oncogene does not encode the extracellular ligand-binding domain or a short C-terminal region which contains the main sites of self-phosphorylation [Downward et al., Nature, 307: 521-527 (1984)].
EGFr has also been shown to have a high degree of structural and sequence homology with the c-erbB-2 protein encoded by the c-erbB-2 oncogene. As is the EGFr, the c-erbB-2 protein is a transmembrane glycoprotein that has an extracellular domain, a transmembrane domain that includes two cysteine-rich repeat clusters, and a cytoplasmic kinase domain. However, the c-erbB-2 protein has a molecular weight of 185 kd and an unidentified ligand [Semba et al., PNAS, 82: 6497-6501 (1985)]. The EGFr and c-erbB-2 protein are encoded by different genes.
Ligand binding to the EGFr is one factor involved in the control of cellular proliferation. Binding of EGF to the ectodomain of EGFr results in a cascade of events beginning with the autophosphorylation of the receptor, followed by aggregation and internalization of EGFrs and ultimately cellular proliferation. It has been demonstrated that ligand binding to the EGFr promotes phosphorylation of the c-erbB-2 protein [King et al., EMBO J., 7 (6): 1647-1651 (1988)]; that observation suggests a mechanism for communication between receptors that could affect control of cell growth. Overexpression of EGFrs, as occurs in A431 cells, can augment cell growth because of increased formation of active ligand/receptor complexes.
Cellular proliferation exhibited by transformed cells may be due to an autocrine mechanism wherein transformed cells both secrete mitogenic growth factors and respond to them in an uncontrolled fashion [Sporn and Todaro, N. Engl. J. Med., 303: 878-880 (1980)]. Thus, the expression levels of growth factors and their receptors are considered important factors in the control of growth.
Gene amplification has been demonstrated to be one mechanism for aberrant EGFr expression in A431 cells and in some glioblastoma multiforme tumors but not in all human tumors [Thompson and Gill, Cancer Surveys, 4(4): 768-788 (1985)]. Studies with tumor cell lines in vitro have shown a direct relationship between the levels of activated EGFr and cellular proliferation. Further, the growth rate of tumors in nude mice was shown to be directly related to the EGFr levels, that is, the most rapidly growing tumors displayed elevated EGFr levels [Thompson and Gill, id.]. Therefore, evidence indicates that measurement of EGFr levels could be important in cancer management.
EGF receptors (EGFrs) can be detected in a variety of cells either by measurement of EGF binding [reviewed in Adamson and Rees, Mol. Cell Biochem., 34:129-152 (1981)], by cross-linking of labeled EGF to its receptor [reviewed in Linsley et al., in Membrane Receptors (eds. Jacobs and Cuatrecasas), vol. B11: 87-113 (Chapman J. Hall, London and New York 1981)], or through the use of monoclonal antibodies [Schreiber et al., PNAS (USA), 78: 7535-7539 (1981); Waterfield et al., J. Cell Biochem., 20: 149-161 (1982); Kawamoto et al., PNAS (USA), 80: 1337-1341 (1983); Richert et al., J. Biol. Chem., 258: 8902-8907 (1983); Gregoriou and Rees, Cell Biol. Int. Reports, 7: 539-540 (1983); and Schlessinger et al., in Receptors and recognition: antibodies against receptors (ed. Greaves) (Chapman and Hall, London, 1985)].
Several groups have investigated the expression of EGFr in a variety of tumors using quantitative as well as semiquantitative immunohistochemical methods. The types of tumors investigated include gynecological [Bauknecht et al., Gynecologic Oncology, 29(2): 147-157 (February 1988); Gullick et al., Cancer Res., 46: 285-292 (1986)]; lung [Berger et al., J. Pathol., 152: 297-307 (1987)]; bladder [Smith et al., Cancer Res., 49: 5810-5815 (1989); Neal et al., Lancet, 366-368 (1985)]; and breast carcinomas [Fitzpatrick et al., Cancer Res., 44: 3448-3453 (1984); Nicholson et al., Int. J. Cancer, 42: 36-41 (1988); and Nicholson et al., Lancet, 182-185 (1989)]. Such studies almost exclusively rely upon radioligand binding methodology for quantitating EGFr in tissue samples.
The most extensive correlations of EGFr expression with clinical data have been carried out in studies with breast cancer patients [Nicholson et al., Int. J. Cancer, 42: 36-41 (1988); Sainsbury et al., Lancet, 1398-1402 (1987)]. In several studies with up to 246 patients, it was demonstrated that EGFr is a highly significant marker of poor prognosis for breast cancer [Sainsbury et al., id.]. It is considered to be the most important variable in predicting relapse-free and overall survival in lymph node-negative patients, and to be the second most important variable, after nodal status, in lymph node-positive patients. In general, EGFr positive tumors are larger and occur in a higher proportion of patients with lymph node involvement.
Further, in breast cancer studies, EGFr has been determined to be as good an indicator as the estrogen receptor (ER) in predicting the response to endocrine therapy [Nicholson et al., Lancet, 182-185 (1989)]. Many ER positive patients don't respond to endocrine therapy, whereas a small percentage of ER negative patients do respond. EGFr positive tumors do not respond to endocrine therapy upon relapse, regardless of ER status, and EGFr positivity reduces the response rate even in ER positive tumors.
Studies have demonstrated a survival advantage for women that have ER positive tumors; most of such tumors are EGFr negative. ER negative tumors are divided into EGFr positive and negative groups, wherein the ER negative/EGFr positive group have a poor prognosis, and the ER negative/EGFr negative group have as good a relapse-free survival rate as the ER positive group. Thus, the measurement of EGFr levels is a useful adjunct to ER level measurement in breast cancer management.
Ovarian, cervical, vulval and endometrial tumors also overexpress EGFr. An association between EGFr levels and prognosis similar to that for breast cancer has been shown for endometrial cancer; however, increased EGFr levels associated with ovarian cancer correlate with a high response rate to chemotherapy [Bauknecht et al., supra]. EGFr expression and gene amplification has been more frequently observed among squamous cell carcinomas of the cervix and lung than in other types of cancer; however, correlation with tumor aggressiveness in such cancers has not been proven [Gullick et al., supra]. Quantitative studies with bladder tumors have shown that elevated EGFr levels are indicative of the most invasive tumors [Smith et al., supra; Neal et all, supra].
Waterfield et al., Cell Biol. Internat'l Reports, 7 (7):535-537 (July 1983), reported the generation of a series of monoclonal antibodies raised against the human cervical carcinoma cell line A431. Hapgood et al., PNAS (USA), 80 (21):6451-6455 (November 1983), describes the generation of different monoclonal antibodies against various domains of the EGFr. A IgM monoclonal is reported to bind to a domain close to the combining site for EGF, and another IgG monoclonal antibody is described as binding to an antigenic determinant that is remote from the combining site for EGF (p. 6454, lines 16-23).
Ullrich et al., Nature, 309 (5967):418-425 (May 31, 1984), provides the complete 1,210 amino acid sequence of the EGFr precursor deduced from cDNA clones derived from placental and A431 carcinoma cells. The receptor gene is therein noted to be amplified and rearranged in A431 cells, generating a truncated 2.8-kilobase (kb) mRNA which encodes only the extracellular EGFr binding domain. [See also, Merlino, Science, 229: 417-419 (Apr. 27, 1984); Xu et al., Nature, 309: 806-810 (Jun. 18, 1984); and Lin et al., Science, 224: 843-848 (1984)]. Ullrich et al. note at page 425 that since the A431 cell line and the many variants thereof have "a multitude of defects exemplified by its 78 chromosomes, it is clearly best to regard this cell line with caution when exploring the mechanism of action of EGF or in characterizing changes which relate in a meaningful way to neoplasia."
Gullick et al. in Anal. Biochem., 141: 253-261 (1984) describe a radioimmunoassay for solubilized EGFr. Gullick et al. in the Proc. R. Soc. Lond, B 226: 127-134 (1985) report on the production of polyclonal and monoclonal antibodies to selected synthetic peptides from the EGFr.
Gullick et al. in Cancer Res., 46: 285-292 (January 1986) describe the properties of two monoclonal antibodies produced to a synthetic peptide consisting of residues 985 to 996 from the cytoplasmic domain of EGFr. Further therein, samples of human tumors were solubilized in detergent and analyzed for the presence of functional EGFrs by autophosphorylation and immunoprecipitation using a monoclonal antibody "which binds to the native folded external domain of the human and rat EGF receptors".
Waterfield et al. [U.S. Pat. No. 4,933,294 filed Dec. 2, 1985 and issued Jun. 12, 1990, entitled "Method of Detecting Truncated Growth Factor Receptors"] disclose that neoplastic and other diseases can be diagnosed by assaying a human test sample, for example body fluid, tissue or cultured tumor explant calls, for structurally altered or abnormally expressed growth factor receptors. Claim 1 from which all the other claims therein depends reads "[a] method of diagnosis for the detection of abnormalities in mammalian cell growth comprising obtaining a test sample from a human and assaying the sample of a truncated epidermal growth factor receptor having at least a portion of its mature amino terminus deleted, and correlating detection of said truncated growth factor receptor with abnormal growth control in mammalian cells."
Waterfield et al.'s International Publication No. WO 85/03357 (published Aug. 1, 1985) entitled "Improvements Relating to Growth Factors" is a foreign counterpart application to U.S. Pat. No. 4,933,294 (discussed immediately above). Claim 46 of that published application reads "[a] method of human diagnosis comprising assaying a human body fluid sample for EGF receptor." [That application was also published as European Patent Application No. 171,407 on Feb. 19, 1986.]
Cline et al. [U.S. Pat. No. 4,699,877 (filed Nov. 20, 1984 and issued Oct. 13, 1987)] describes methods and compositions for detecting the presence of tumors, wherein a physiological sample is assayed for the expression product of an oncogene.
Weber and Gill, Science, 224: 294 (1984), reported that human epidermoid carcinoma A431 cells in culture produce a soluble 105 kilodalton (kd) protein which they determined to be related to the cell surface domain of the EGFr. They further determined that the soluble receptor 105 kd protein was not derived from the membrane-bound intact receptor but was separately produced by the cell. [See also, Mayes and Waterfield, EMBO J., 3: 531-537 (1984); and Cooper et al., J. Virol., 48:752-764 (1983).]
Four years later, Weber in an abstract entitled "Truncated EGF receptor: functional aspects of its secretion," [Acta Endocrino Logica, 117 (Suppl. 287): 47 (No. 55) (1988)] reported that two products of the EGFr gene "are expressed in human A431 epidermoid carcinoma cells: the `normal` 170 kDa receptor which is inserted into the membrane, and a 100 kDa EGF receptor-related protein (ERRP) which is secreted . . . " That ERRP was described as corresponding to the extracellular domain of the EGFr. Weber further reported finding ERRP in another line of tumor cells, and that when solid A431 tumors were grown on athymic mice, that ERRP appeared in the blood of the mice and could be measured in the serum.
Co-pending U.S. patent application Ser. No. 389,920 (filed Aug. 4, 1989) discloses assays for detecting the external domain glycoprotein (gp 75) encoded by the c-erbB-2 oncogene or parts thereof in the body fluids of mammals. The application claims methods prognostic and diagnostic for neoplastic disease based upon such assays.
As indicated above, several methods have been used to detect EGFr levels in tumor tissues. There are, however, many cases in which tissue is not readily available or in which it is not desirable or not possible to withdraw tissue from tumors. Therefore, there is a need in the medical art for rapid, accurate diagnostic tests that are convenient and non-traumatic to patients. The invention disclosed herein meets said need by providing for non-invasive diagnostic/prognostic assays to detect and/or quantitate in mammalian body fluids, preferably serum, a portion of the EGFr which comprises substantially the ectodomain of EGFr.